High-density lipoprotein cholesterol (HDL-c) is an especially promising candidate for therapeutic intervention against coronary heart disease (CHD). Although, absolute levels of HDL-c correlate inversely with the CHD risk, there is growing evidence that cholesterol flux in HDL particles from peripheral tissues to the liver - i.e., reverse cholesterol transport (RCT) - is a more important atheroprotective factor. Recent research shows that different HDL particle species stimulate RCT to varying extents. The origins of particle heterogeneity are unclear. Some particle speciation is already evident in nascent HDL. Nascent HDL is assembled from cellular lipids and extracellular apolipoprotein AI (apoAI) through a process mediated by ATP-binding cassette transporter A1 (ABCA1). In Specific Aim 1, we propose to test the hypothesis that localization of ABCA1 in different microenvironments of the plasma membrane is responsible for nascent HDL particle heterogeneity. Lipid composition of the plasma membrane and putative localization of ABCA1 to different plasma membrane domains will be manipulated to determine what effects these manipulations exert on the nascent HDL population. ApoAI binding protects ABCA1 from degradation and promotes nascent HDL particle formation in a feed-forward regulatory loop. In Specific Aim 2, we propose two primary approaches to identify putative apoAI binding sites on ABCA1. In one approach, ABCA1 and apoAI will be cross-linked and then ABCA1-apoAI complexes will be analyzed using mass spectrometry. In the second approach, computationally selected candidate ABCA1 regions will be chemically synthesized and tested for binding to apoAI using surface plasmon resonance. The regions of ABCA1 identified with the two approaches will be validated using mutagenic analyses and binding completion assays. The knowledge gained from this project will aid in design of novel therapies to stimulate RCT and maximize production of the most atheroprotective HDL species. PUBLIC HEALTH RELEVANCE: Coronary heart disease is a leading cause of death in the US and the world. Present therapies that focus on lowering levels of bad (LDL) cholesterol reduce but do not completely eliminate coronary heart disease occurrences. The proposed research should facilitate development of supplemental therapies that exploit heart disease reducing properties of good (HDL) cholesterol.